Magneto-motive ultrasound detection of magnetic nanoparticles

ABSTRACT

Provided herein are systems, methods and compositions for the use of ultrasound for detection of cells and nanoparticles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation from U.S. application Ser. No.12/172,592, filed Jul. 14, 2008, which will issue as U.S. Pat. No.8,454,511 on Jun. 4, 2013, and claims priority to U.S. provisionalapplication Ser. No. 60/949,460, filed Jul. 12, 2007, and thisapplication is a continuation-in part of U.S. application Ser. No.11/784,477, filed Apr. 6, 2007, now U.S. Pat. No. 7,801,590, which is acontinuation-in-part of U.S. application Ser. No. 11/441,824, filed May26, 2006, now U.S. Pat. No. 7,983,737, which claims the benefit of U.S.provisional application Ser. No. 60/685,559, filed on May 27, 2005; andthe application is also a continuation-in-part of U.S. application Ser.No. 11/620,562, filed on Jan. 5, 2007, now U.S. Pat. No. 8,162,834,which is a continuation-in-part of U.S. application Ser. No. 11/550,771,filed on Oct. 18, 2006, now U.S. Pat. No. 8,036,732. The aforementionedapplications are herein incorporated by reference in their entirety.

BACKGROUND

Ultrasound is a broadly used tool in medical imaging and it has severaladvantages over other imaging techniques such as MRI and computedtomography (“CT”). Ultrasound is a real-time, nonionizing, costeffective, portable, and widely available imaging modality. Ultrasoundcontrast agents have enabled researchers to expand their investigationsinto molecular scales, contributing to increased contrast enhancementand also contrast-specific imaging. The most common ultrasound contrastagents, composed of specific gaseous microbubbles in a core shell, havebeen investigated to enhance contrast in ultrasound medicine. Comparedto the surrounding tissue, microbubbles present a large acousticimpedance mismatch in tissues and thus produce a strong backscatteredsound signal. In addition, microbubbles are used in harmonic andsubharmonic imaging to improve the subjective image quality. Althoughmicrobubbles play an important role in increasing the enhancement of thediagnostic potential of ultrasound imaging, the micron-sized bubbleshave limited use in molecular imaging because these agents are too largeto pass through the pulmonary and systemic capillary bed. Moreover,microbubbles are unstable, have a short blood half-life and a propensityto fracture and collapse when exposed to ultrasound waves. Furthermore,microbubbles need sufficient acoustic pressure to increase contrast.These features have limited the use of microbubbles in ultrasoundmolecular imaging. To overcome the size effects and increase theefficacy of enhancement imaging, perfluorocarbon emulsion nanoparticles(“PFC”), approximately 250 nm in diameter, were reported as analternative ultrasound contrast agent. Nanometer-sized ultrasoundcontrast agents may penetrate the large capillary bed, but penetrationto vascular targets and extravasations through tight capillary porescould be inhibited due to their relatively large size. Unfortunately,PFC ultrasound contrast agents produced a smaller acoustic impedancemismatch, a weaker ultrasound reflected signal, and create less contrastenhancement of echogenic images than gaseous microbubbles.

Superparamagnetic iron oxide (“SPIO”) nanoparticles have been wellestablished over the past decade as a contrast enhancement for MRIimaging. Early studies demonstrated that SPIO nanoparticles can improvethe detection of liver metastases in patients. After SPIO nanoparticleshave been administrated intravenously, tissue-based macrophages (Kupffercells) in the body take up SPIO nanoparticles through the reticuloendothelial system (“RES”) including bone marrow, hepatic lesions, lymphnode metastases in cancer, and spleen. Compared to conventionalultrasound contrast agents, magnetically activated SPIO nanoparticleshave several advantages, including small size, strong magneticsusceptibility, and bio-safety. These nanoparticles (μ20 nm core size)allow transport through the microvasculature and enable passage throughthe endothelium while retaining their super paramagnetic properties.Since SPIO nanoparticles for tissue-specific MRI contrast agents wereapproved by the FDA in 1996, to date, these magnetic nanoparticles havebeen used in various clinical applications without safety concernsassociated with alternative contrast agents.

The present application improves ultrasound imaging.

SUMMARY OF THE INVENTION

Provided herein are systems, methods and compositions for the detectionof cells using ultrasound using nanoparticles.

The methods, systems, and apparatuses are set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the methods, apparatuses,and systems. The advantages of the methods, apparatuses, and systemswill be realized and attained by means of the elements and combinationsparticularly pointed out in the appended claims. It is to be understoodthat both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof the methods, apparatuses, and systems, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the setup with the magnetic fieldgenerator and conical iron core inside the solenoid.

FIG. 2A is a schematic diagram showing exemplary multifunctionalnanoparticles (MONs) with an iron core for magnetic properties, a goldcoating to tune wavelength absorption to 700 nm (above competing plaquecomponents such as hemoglobin), and absorbed aminodextran coating forselective macrophage uptake.

FIG. 2B is a TEM of ferromagnetic nanoparticles in absence of anexternal magnetic field in lattice form.

FIG. 3A is a schematic diagram showing exemplary multifunctionalnanoparticles (MONs) with an aminodextran outer shell adsorbed on aninner gold shell and FIG. 3B is the molecular structure of aminodextran.

FIG. 4 is a schematic diagram of the setup with the magnetic fieldgenerator and conical iron core inside the solenoid.

FIGS. 5( a)-(e) are ultrasound grayscale images; FIGS. 5( f)-(j) arecolor power Doppler images; and FIGS. 5( k)-(o) M-mode images of liverswith different SPIO doses (1.5, 1.0, 0.5, and 0.1 mmol Fe/kg and controlliver).

FIGS. 6( a)-(j) are graphs of the Doppler shift measured in liverspecimens with different SPIO doses (1.5, 1.0, 0.5, and 0.1 mmol Fe/kg)using swept frequency input ((a)-(e)) and 1 Hz sinusoidal input((f)-(j)).

FIGS. 7( a)-(f) is the Doppler shift from liver specimens (1.0 mmolFe/kg) with constant (8 Vpp) amplitude sinusoidal input.

FIG. 8A is a graph of the peak frequency shift in livers with differentSPIO doses (1.5, 1.0, 0.5, and 0.1 mmol Fe/kg and control) when applyinga swept frequency input (1-10 Hz) with different input voltages (2-10Vpp); and FIG. 8B is a graph showing the magnetic flux density of theswept frequency input signal versus input peak-to-peak voltage.

FIGS. 9A-9B are histological cross-section of iron-laden liver withPrussian blue stain, where 9A is the control specimen and 9B is aspecimen with a high concentration of iron (1.5 mmol Fe/kg body weight);and FIGS. 9A-9B are observed (magnification: 20×), where the blue colorregions are iron oxide nanoparticles engulfed by liver-based macrophageKupffer cells.

FIG. 10 is a schematic diagram of the Magneto-Motive Ultrasound probefor intravascular techniques.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are methods, compositions and apparatuses for detectinga cell and/or a metallic composition using ultrasound and optionallykilling the cell. By a “cell” is meant one or more cell of, or derivedfrom, a living organism or subject. The cell or cells can be locatedwithin a subject or can be located ex vivo. The disclosed methods,compositions and apparatuses for detecting a cell and/or a metalliccomposition are described herein variously by reference to cell(s),composition(s) and/or metallic composition(s). It will be understoodthat description of various aspects of the disclosed methods,compositions and apparatuses by reference to one or a subset of cell(s),composition(s) or metallic composition(s) constitutes description ofthat aspect of the disclosed methods, compositions and apparatuses tothe non-referenced cell(s), composition(s) and metallic composition(s),unless the context clearly indicates otherwise.

In one embodiment, the method for detecting a cell comprises applying amagnetic field to the cell. A cell can comprise a cellular membrane anda metallic composition. Optionally, the metallic composition is ametallic nanoparticle that was administered to the subject or otherwisebrought into contact with the cell. In another exemplary method fordetecting a cell by ultrasound, light energy can cause a change in thecell. For example, a pulsed laser can be used to cause movement of themetallic composition comprised by the cell.

The metallic composition can be located within the cell, including inthe cell's cellular membrane, or on the outside of the cell. If themetallic composition is a metallic nanoparticle located on the outsideof the cell, it can be connected or targeted to the exterior surface ofthe cell's cellular membrane. Exemplary methods of targeting orconnecting a metallic composition to a cell are described herein.

Generally speaking, a system and method for detecting nanoparticles byMagneto-Motive Ultrasound (“MM-US”) is described herein. By combining ahigh strength magnetic field to excite iron-laden tissue, ultrasounddetects the detectable internal strain field or induced tissue motion,as disclosed by inventors in Nanotechnology 17: 4183-4190 (2006), hereinincorporated by reference. As shown in FIG. 1, the MM-US system 100comprises an ultrasound beam 70, metallic compositions 60, and amagnetic field generator 90. The magnetic field generator 90 includes asolenoid coil 82 (Ledex 4EF) with a conical iron-ferrite core 84 at thecenter driven by a current amplifier 86, a signal generator 92 (HP33120A, Hewlett Packard Inc., USA), and a regulated DC power supply 88.The magnetic field generator 90 can be placed near a tissue or cell 62containing nanoparticles 60 during MM-US imaging. The combination of theferrite core 84 and solenoid coil 82 using a high power operationincreases the magnetic field substantially and focuses the magneticfield strength (B_(max)=2 T). The magnetic force on the metalliccomposition 60 is varied by applying a sinusoidal current to thesolenoid 82 containing a conical iron-ferrite core 84 to generate adetectable internal strain field on the cell 62. A detectable internalstrain field may be any microscale displacement of the nanoparticle ornanoparticle-tissue conglomerates, which is detected by ultrasound.

Ultrasound imaging systems can be equipped with a 38 mm aperture,broadband (5-10 MHz) linear array transducer. Cells can be imaged incolor power Doppler, power Doppler, M-mode and B-scan modes. B-scansonogram images, also called the grayscale mode, are the typicalultrasound method to monitor or examine the human body usingbackscattering of acoustic waves. M-mode ultrasound employs a sequenceof scans at a fixed ultrasound beam over a given time period. M-mode isused for visualizing rapidly moving subjects, such as heart valves.Compared to conventional B-scan images, Doppler ultrasound is used toassess changes in the frequency of reflected acoustic waves. Color powerDoppler converts reflected acoustic waves that are Doppler shifted intocolors that overlay the conventional B-scan images and can indicate thespeed and direction of moving objects. Power Doppler ultrasound is mostcommonly used to evaluate moving objects and has higher sensitivity thanthe color power Doppler mode. The gain of the color power and Dopplerimaging mode can be manually adjusted to suppress the background noise.If the settings of the ultrasound instrumentation remain unchanged,objective comparisons of each can be made.

Alternatively, the MM-US imaged nanoparticles can then be affected withan energy capable of heating the metallic composition, wherein theheating is sufficient to kill, lyse the cell, or lethally injure thedetected cell. Other non-limiting examples of energy that can be appliedto kill a cell include any energy that can move the nanoparticle of thecell. Movement of the particle can be used to heat the particle to asufficient degree to kill or lyse the cell. For example, magnetic,light, or sonic energy can be used.

An effective cell killing protocol can vary with such factors as theparticular cell being killed, the tissue in proximity to the cell, thetype and composition and characteristics of the particle, the number ofparticles, the type of pathology being treated, the duration of thetreatment, characteristics of the treatment (i.e. wavelength, fluence,pulse duration and number of pulses) the nature of concurrent therapy(if any), the type of energy being applied to contact the particle ofthe cell, the properties of the surrounding media or amount of water. Aneffective killing protocol can be readily determined by one of ordinaryskill in the art using routine experimentation.

After detecting a cell by MM-US, for example a macrophage associatedwith vulnerable plaque, the system can further comprise an energy sourcefor heating the metallic particle or composition. The source can provideenergy for heating the composition that is sufficient to kill orlethally injure the detected cell. In some exemplary aspects, the systemcan further comprise an energy source for causing a non-lethal change inthe cell. For example, the energy source for causing a non-lethal changein the cell can produce a magnetic field. The energy source for causinga non-lethal change in the cell can also produce light or sound. Theenergy source for causing the non-lethal change in the cell and theenergy source for heating the metallic particle can be of the same type.For example, each energy source can generate and/or transmit soundenergy. In some exemplary aspects, the energy source for causing thenon-lethal change in the cell and the energy source for heating themetallic particle are the same source. In other exemplary aspects, theenergy source for causing the non-lethal change in the cell and theenergy source for heating the metallic particle are different sourcesand/or different types of energy. For example, the energy sources forcausing the non-lethal change can generates and/or transmits magneticfield energy and the energy source for causing the heating can generateand/or transmits light energy. Thus, in some exemplary aspects thesystems described herein can comprise at least three separate sources ofenergy. One source of energy can be the magnetic energy used for MM-USimaging, as described previously. Such magnetic energy can be referredto as imaging magnetic energy. A second source can be used to produceenergy to cause heating of the metallic composition comprised by thecell to kill or lethally injure the cell. Such sources can increase thetemperature of a metallic particle in the cell. For example, any sourcethat can increase the particle temperature can be used. Exemplarysources include light energy sources and magnetic force generators thatcan cause an increase in temperature of the particle by inducingmovement of the particle. Such sources to cause lethal changes in a cellcan comprise magnetic fields, light, sound and any other energy that cancause lethal changes to a cell. The magnetic fields may also beoscillating or alternating or a DC magnetic field.

The detectable internal strain field can be generated in a cell when ametallic composition, including a metallic nanoparticle, is under theaction of an external force. The internal strain field can be detectedusing ultrasound imaging. The external force may be provided by theapplication of an external magnetic flux density (B). An external forceon the nanoparticle may be induced by the interaction of the externalmagnetic flux density (B) with an induced magnetic moment in thenanoparticle (paramagnetic or diamagnetic) or with a permanent magneticmoment (ferromagnetic) in the nanoparticle. Action of the external forceon each metallic composition can produce movement of the metalliccomposition (z_(np)(t)) that produces a change in the cellular membranetension level or an internal strain field within a cell. Action of aforce on each metallic composition in a cell or tissues produces amovement of the metallic composition (z_(np)(t)). Movement of themetallic composition can be along the z-direction. The metalliccomposition can also have movement in any direction that can be writtenas vector displacement, u_(np)(r_(o)) for a metallic compositionpositioned at r_(o). Metallic composition displacement u_(np)(r_(o)) canproduce a displacement field (u(r, r_(o))) in the proteins in the cellcontaining the metallic composition and surrounding cells. In the caseof a homogeneous elastic media, the displacement field (u(r, ro)) can becomputed by tissue biomechanical methods, which are well known in theart. The displacement field (u(r, ro)) produced by a metalliccomposition positioned at r_(o) can induce an internal strain field thatis determined by change in the displacement field along a particulardirection. The strain field (ε_(ij)(r, r_(o))) is a tensor quantity andis given by,

${ɛ\left( {r,r_{0}} \right)} = \frac{\partial{u\left( {r,r_{0}} \right)}}{\partial x_{j}}$

where u_(i)(r, r_(o)) is the i′ th component of the displacement fieldand x_(j) is the j^(th) coordinate direction. For example, when j=3, x₃is the z-direction. The internal strain field in a cell due to allmetallic compositions in the cell and surrounding cells is asuperposition of the strain fields due to each metallic composition. Adetectable change in a cell can also be caused with light energy. Forexample, pulsed laser light can be applied to contact a metallicparticle comprised by a cell including in a cell either naturallyoccurring or administered exogenously. The application of light energycan cause a detectable change due to a change in optical refractive andthermal elastic expansion. The light energy can also cause motion of thecell, particle, or tissues proximate to the cell for detection byultrasound, light is absorbed by the nanoparticle and generates anacoustic wave that is detected by an acoustic transducer. Alternatively,sound energy can motion of the cell, particle, or tissues proximate tothe cell for detection by ultrasound.

Metallic Nanoparticles

The metallic composition can comprise a plurality of metallicnanoparticles. The nanoparticles can be substantially spherical in shapeand can have a diameter from about 0.1 nanometers (nm) to about 1000.0nm. The nanoparticles are not, however, limited to being spherical inshape. Thus, the nanoparticles are asymmetrical in shape. If thenanoparticles are asymmetrical in shape, the largest cross sectionaldimension of the nanoparticles can be from about 0.1 nanometers (nm) toabout 1000.0 nm in length. The metallic composition can comprise metalhaving non-zero magnetic susceptibility or zero magnetic susceptibilityor combinations of non-zero and zero magnetic susceptibility metals. Inaddition, the metallic composition may be ferromagnetic and therebycontain a permanent magnetic moment. Thus, if the composition comprisesnanoparticles, the nanoparticles can all have a non-zero magneticsusceptibility or a zero magnetic susceptibility or a combination ofparticles having a non-zero magnetic susceptibility and a zero magneticsusceptibility. Metallic compositions having a non-zero magneticsusceptibility can comprise a material selected from the groupconsisting of iron oxide, iron, cobalt, nickel, chromium andcombinations thereof. The metallic compositions can comprise metalhaving non-zero electrical conductivity or zero electrical conductivityor combinations of non-zero and zero electrical conductivity metals.Also provided is a method for detecting a composition, the methodwherein the composition comprises a magnetic or paramagnetic material.Any magnetic or paramagnetic material, whether metallic or non-metallic,can be used in the described methods or with the described systems. Inthis regard, any material can be used that can cause a change in a cellor can be detected using ultrasound when contacted with an appliedmagnetic field. Similarly, nonmetallic, non-magnetic particles can beused to cause a change in a cell or can be detected using ultrasoundwhen contacted with an applied magnetic field using the methods andsystems described herein. Also, as described herein, a pulsed lightsource can be applied to a cell, typically in pulse duration from 5fs-50 ns and light from a pulsed laser source is absorbed by thenanoparticle and generates an acoustic wave that is detected by anacoustic transducer.

The systems, apparatuses and methods can be practiced using metalliccompositions without magnetic susceptibility. When using metalliccompositions without magnetic susceptibility, or when using compoundshaving a non-zero magnetic susceptibility, an electrical eddy currentcan be induced in the composition by a changing magnetic flux density.

To induce an eddy current in a metallic composition a first time-varyingmagnetic field can be applied to a cell. The first magnetic field caninteract with a metallic composition within or external to the cell toinduce an electrical eddy current within the metallic composition. Asecond magnetic field can be applied to the cell that interacts with theinduced eddy current to cause a change in the cell. The cell can bedetected by detecting the change in the cell caused by the interactionof the second magnetic field with eddy current using an ultrasoundmodality. Exemplary changes in the cell caused by the interaction of thesecond magnetic field with the eddy current include movement of thecell, movement of the metallic composition, a change in the cellularmembrane tension level, and a change in the internal strain field of thecell.

Thus, a metallic composition or a nanoparticle that does not have asignificant magnetic permeability can be used. For example, althoughgold nanoparticles do not have significant magnetic permeability manytarget-specific molecular agents (e.g., antibodies) can be conjugated tothe nanoparticle surface. When using a high-conductivity particle fordetection, a magnetic dipole can be induced in the particle by exposingto a time-varying magnetic field (B(t)).

The time-varying magnetic field (B(t)) can cause an electromotive forceor potential in the particle that can induce a volumetric and surfaceelectric eddy-current in the high conductivity nanoparticle. Exemplarycircuitry for a magnetic pulser that can be used to produce an eddycurrent is described in G H Schroder, Fast pulsed magnet systems,Handbook of Accelerator Physics and Engineering, A. Chao and M. Tinger,Eds. 1998 or in IEEE transactions on instrumentation and measurement,VOL. 54, NO. 6, December 2005, pp 2481-2485, which is incorporatedherein by reference for the circuitry and methods described therein.

The eddy-current can produce time-varying magnetic moment that caninteract with a second applied magnetic field (B). The inducededdy-current in the high-conductivity nanoparticle or metalliccomposition and the second applied magnetic field can interact toproduce a torque or twist and or force on the nanoparticle or metalliccomposition. The induced torque can twist or move the nanoparticle thatis mechanically linked to a target in the cell (e.g., the membrane) orlocated inside the cell. The displacement or twisting motion of thenanoparticle can modify the internal strain field of the cell(surrounding cells and tissue) which can be detected using phasesensitive optical coherence tomography. In this approach,phase-sensitive data can be recorded before and after application of afirst field to induce an eddy current and block correlation algorithmscan be used to compute the depth resolved strain field in the tissueresulting from the motion of the nanoparticle or metallic composition.

Ferromagnetic nanoparticles have a permanent magnetic moment, i.e. theferromagnetic nanoparticles are magnetized. Ferromagnetic particles maybe synthesized from iron with a minimum diameter is about 20-30 nm orCobalt with a minimum diameter is about 10-12 nm. In additionferromagnetic particles may be coated with gold as the superparamagneticparticles are coated with gold.

In this case the externally applied magnetic flux density (B) interactswith the magnetic moment in the nanoparticle and can produce eitherlinear force or torque that induces an internal strain field in thetissue.

In exemplary embodiments, large magnetic fields can be generated by lowtemperature superconducting magnets. These magnets need only be“charged” once, maintained at a low temperature and do not require anexternal current to maintain the magnetic field.

A metallic composition can be administered to the subject.Administration of exogenous metallic compositions, for example, metallicnanoparticles is described in greater detail below. Optionally, the cellcan be located within a subject and the metallic composition can beadministered to the subject. Optionally, the cell can be a macrophageand at least one metallic nanoparticle can be located within themacrophage or can be connected to the macrophage. The macrophage can belocated in an atherosclerotic plaque within the subject. The macrophagecan also be located within the eye of the subject.

In the methods described herein, a nanoparticle comprising a materialwith non-zero magnetic susceptibility can be positionally moved in vivo,in vitro, or ex vivo by an applied magnetic field. A material ofnon-zero magnetic susceptibility can include a variety of materials. Forexample, the nanoparticle can comprise any physiologically tolerablemagnetic material or combinations thereof. The term magnetic materialcan optionally include any material displaying ferromagnetic,paramagnetic or superparamagnetic properties. For example, thenanoparticles can comprise a material selected from the group consistingof iron oxide, iron, cobalt, nickel, and chromium. Metallic compositionsas described throughout, including administered nanoparticles, can bemagnetic. Optionally, a nanoparticle comprises iron oxide. When ananoparticle comprises metal or magnetic materials, it can be movedwhile in the subject using an internally or externally applied magneticfield, as described below. Any relevant metal with non-zero magneticsusceptibility or combinations thereof can be used. Many useable metalsare known in the art; however, any metal displaying the desiredcharacteristics can be used. Nanoparticles can also comprise acombination of a material with a non-zero magnetic susceptibility and amaterial with a lower or zero magnetic susceptibility. For example, goldcan be combined with higher magnetic susceptibility materials (e.g.,iron). For example, gold coated iron can be used that still have apermanent magnetic moment, i.e. ferromagnetic. Nanoparticles can alsoinclude a polymer coating or other coating materials alone or incombination, which are electrically conductive or contain metallic atomswith a magnetic moment. Alternatively, such polymers or coatingmaterials can be used to attach targeting ligands, including but notlimited to antibodies, as described below. When used in vivo, anadministered nanoparticle can be physiologically tolerated by thesubject, which can be readily determined by one skilled in the art.

Nanoparticles can be solid, hollow or partially hollow and can bespherical or asymmetrical in shape. Optionally, the cross section of anasymmetric nanoparticle is oval or elliptical. As one of skill in theart will appreciate, however, other asymmetric shapes can be used. Inone example, the particle can be shaped like a bacterium. A bacteriumshaped particle can be used to increase the likelihood of engulfment ofthe particle by a macrophage. The nanoparticles can comprise shelled ormulti-shelled nanoparticles. Each shell layer can be metal. Amulti-shelled particle can also have one or more layers that arenon-metallic. For example, the particles can be coated with sugar,polysaccharide, protein, peptide, polypeptide, amino acid, nucleic acid,and portions or fragments of each of these coating compositions.Moreover, each coating composition or portion thereof, or metalcomposition can fully or partially surround any other portion of aparticle.

One exemplary particle comprises iron oxide and gold. The iron oxide canform a core that is surrounded partially or fully by a gold layer.Dextran can be applied to the gold layer to comprise a particle of iron,gold and dextran. Other exemplary layers can also be used. For example,a metallic core can selected based on its magnetic properties so that itcan be moved in the subject by an applied magnetic force. A second metallayer can be selected based on its light absorption properties and thelight absorptive characteristics of the tissue or media where theparticle is located. For example, gold can be used to enhance theabsorption of near infrared light by the particle in the cell. Exemplarycombinations of materials for particles that can be moved by an appliedmagnetic force and can absorb light more than proximate tissue or cellsof the subject can be selected using the principles of photothermolysis,known in the art and described below.

Exemplary nanoparticles are shown in FIGS. 2 and 3. FIG. 2A is aschematic diagram showing exemplary multifunctional MMUS nanoparticlewith an iron core for magnetic properties, a gold coating to tunewavelength absorption to 700 nm (above competing plaque components), andan adsorbed amino-dextran coating for enhanced selective macrophageuptake. FIG. 2B is a Transmission Electron Microscope image offerromagnetic nanoparticles in lattice form. Ferromagnetic nanoparticlesinclude have a minimum size that is required to form a domain, i.e. forIron ferromagnetic nanoparticles the size is about 25 nm in diameter forthe iron core. Second, the ferromagnetic nanoparticles have a permanentmagnetic moment that is represented by a vector. The vector can point inany direction relative to the particle.

FIG. 3A is a schematic diagram showing several exemplary multifunctionalMMUS nanoparticles (MONs) with an amino-dextran outer shell adsorbed onan inner gold shell. As shown in FIG. 3B, additional NH₂ sites on thedextran that are not bound to gold can be used to conjugate smallmolecules such as fluorescein isothiocyanate (“FITC”), or and Glycine toraise the selectivity for macrophage uptake. Particle shape can also bealtered to mimic the rod-like appearance of bacteria to enhancemacrophage uptake, as shown in FIG. 3A.

Shelled or multi-shelled nanoparticles can have targeting ligandsconjugated to the shell material wherein the targeting ligand has anaffinity for or binds to a target site in a subject or ex vivo. Suchshelled or multi-shelled nanoparticles can be made, for example, usingtechniques known in the art, for example, as described in Loo et al.,“Nanoshell-Enabled Photonics-Based Imaging and Therapy of Cancer,” Tech.Cancer Res. and Treatment, (2004) 3(1) 33-40, which is incorporatedherein by reference for the methods taught herein. Further, Oldenburg etal., “Nanoengineering of Optical Resonances,” Chemical Physics Letters(1998) 288, 243-247, is incorporated herein for methods of nanoshellsynthesis.

The methods of detecting cells and compositions using ultrasound cancomprise administering a plurality of metallic nanoparticles to asubject.

Optionally, at least one administered nanoparticle localizes within amacrophage located in the subject. At least one administerednanoparticle can also be optionally configured to localize to a targetsite in the subject.

Localized Nanoparticle to a Target Site

A metallic composition, including a nanoparticle, can be configured tolocalize to a target site within the subject. For example, the metalliccomposition can be configured to localize to a neoplastic cell, to apeptide, to a protein, or to a nucleic acid. Optionally, the target siteis an extracellular domain of a protein. A variety of cell types canalso be targets of the metallic compositions. For example, target cellscan be selected from one or more of a neoplastic cell, a squamous cell,a transitional cell, a basal cell, a muscle cell, an epithelial cell,and a mucosal cell. The target cells can also be located at differentanatomical locations within a subject. For example, the cell can belocated in the subject at an anatomical location selected from the groupconsisting of a lung, bronchus, intestine, stomach, colon, eye, heart,blood vessel, cervix, bladder, urethra, skin, muscle, liver, kidney, andblood.

One or more administered nanoparticle can localize to a desired targetwithin the subject using passive or active targeting mechanisms. Passivetargeting mechanisms take advantage of the subject's inherent defensemechanisms to highlight phagocytic cells naturally responsible forparticle clearance. For example, macrophage rich areas are apathological correlate to an unstable atherosclerotic plaque in asubject. Moreover, administered nanoparticles, for example, smallsuperparamagnetic and ultrasmall superparamagnetic particles of ironoxide, are avidly taken up, or engulfed by, macrophages located inunstable plaques. Thus, through the subject's natural defense mechanism,wherein macrophages accumulate in an unstable atherosclerotic plaque andengulf administered nanoparticles, administered nanoparticles canpassively target the unstable plaque. Similarly, macrophages located inthe eye of a subject can engulf nanoparticles. Such passive targeting ofnanoparticles can be used with the methods and apparatuses describedherein to highlight a plaque's instability or to highlight otheraccumulation of phagocytic cells.

Active targeting mechanisms can refer to the use of ligand-directed,site-specific targeting of nanoparticles. A nanoparticle can beconfigured to localize to a desired target site in a subject using awide variety of targeting ligands including, but not limited to,antibodies, polypeptides, peptides, nucleic acids, and polysaccharides.Such nanoparticles are referred to herein as “targeted nanoparticles.”Targeting ligands or fragments thereof can be used to target ananoparticle to cellular, or other endogenous or exogenous biomarkers inthe subject. Such a biomarkers or “target sites” can include, but arenot limited to, proteins, polypeptides, peptides, polysaccharides,lipids, or antigenic portions thereof, which are expressed within thesubject. When active targeting mechanisms are used to target a cell, thetargeted nanoparticle can be optionally internalized by the targetedcell.

Thus, using the disclosed methods, at least one administerednanoparticle can optionally localize within a macrophage located in thesubject and/or at least one administered targeted nanoparticle canlocalize to a desired target site in the subject.

The methods and apparatuses are not, however, limited to in vivoadministration to a subject. As would be clear to one skilled in theart, nanoparticles, including targeted nanoparticles, can beadministered in vitro to an ex vivo sample with localization of thenanoparticle to a desired target site and subsequent imaging occurringin vitro. Moreover, a composition, including at least one nanoparticlecan be administered to a subject in vivo, and a sample can besubsequently taken from the subject and imaged ex vivo using themethods, systems, and apparatuses described herein.

When using a targeted nanoparticle the target site in vivo or in vitrocan be endogenous or exogenous. The target site can be selected from thegroup consisting of an organ, cell, cell type, blood vessel, thrombus,fibrin and infective agent antigens or portions thereof. Optionally, thetarget site can be a neoplastic cell. The target site can also be anextracellular domain of a protein. Furthermore, the target site can beselected from the group consisting of a lung, bronchus, intestine,stomach, colon, heart, brain, blood vessel, cervix, bladder, urethra,skin, muscle, liver, kidney and blood. The target site can also be acell. For example, a cell can be selected from the group consisting of,but not limited to, a neoplastic cell, a squamous cell, a transitionalcell, a basal cell, a muscle cell, an epithelial cell, a lymphocyte, aleukocyte, a monocyte, a red blood cell, and a mucosal cell.

Thus, targeted nanoparticles can be targeted to a variety of cells, celltypes, antigens (endogenous and exogenous), epitopes, cellular membraneproteins, organs, markers, tumor markers, angiogenesis markers, bloodvessels, thrombus, fibrin, and infective agents. For example, targetednanoparticles can be produced that localize to targets expressed in asubject. Optionally, the target can be a protein, and can be a proteinwith an extracellular or transmembrane domain. Optionally, the targetcan be the extracellular domain of a protein.

Desired targets can be based on, but not limited to, the molecularsignature of various pathologies, organs and/or cells. For example,adhesion molecules such as integrin αvβ3, intercellular adhesionmolecule-1 (I-CAM-1), fibrinogen receptor GPIIb/IIIa and VEGF receptorsare expressed in regions of angiogenesis, inflammation or thrombus.These molecular signatures can be used to localize nanoparticles throughthe use of a targeting ligand. The methods described herein optionallyuse nanoparticles targeted to one or more of VEGFR2, I-CAM-1, αvβ3integrin, αv integrin, fibrinogen receptor GPIIb/IIIa, P-selectin,and/or mucosal vascular adressin cell adhesion molecule-1.

As used, the term “epitope” is meant to include any determinant capableof specific interaction with a targeting ligand as described below.Epitopic determinants can consist of chemically active surface groupingsof molecules such as amino acids or sugar side chains and can havespecific three dimensional structural characteristics, as well asspecific charge characteristics.

Targeting ligands specific for a molecule that is expressed orover-expressed in a cell, tissue, or organ targeted for imaging, such aspre-cancerous, cancerous, neoplastic, or hyperproliferative cells,tissues, or organs, can be used with the nanoparticles described herein.This use can include the in vivo or in vitro imaging, detection, ordiagnosis of pre-cancerous, cancerous, neoplastic or hyperproliferativecells in a tissue or organ. The compositions and methods can be used orprovided in diagnostic kits for use in detecting and diagnosing cancer.

As used herein, a targeted cancer to be imaged, detected or diagnosedcan be selected from, but are not limited to, the group comprisinglymphomas (Hodgkins and non-Hodgkins), B cell lymphoma, T cell lymphoma,myeloid leukemia, leukemias, mycosis fungoides, carcinomas, carcinomasof solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas,gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas,melanomas, adenomas, hypoxic tumors, myelomas, AIDS-related lymphomas orsarcomas, metastatic cancers, bladder cancer, brain cancer, nervoussystem cancer, squamous cell carcinoma of head and neck,neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer,melanoma, squamous cell carcinomas of the mouth, throat, larynx, andlung, colon cancer, cervical cancer, cervical carcinoma, breast cancer,epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer,esophageal carcinoma, head and neck carcinoma, hematopoietic cancers,testicular cancer, colo-rectal cancers, prostatic cancer, or pancreaticcancer.

Pre-cancerous conditions to be imaged, detected or diagnosed include,but are not limited to, cervical and anal dysplasias, other dysplasias,severe dysplasias, hyperplasias, atypical hyperplasias, and neoplasias.As would be clear to one skilled in the art, however, additional cancersand pre-cancerous conditions can be imaged, detected or diagnosed usingthe methods and apparatuses described herein.

Using methods known in the art, and as described herein, targetingligands, such as polyclonal or monoclonal antibodies, can be produced todesired target sites in a subject. Thus, a targeted nanoparticle canfurther comprise an antibody or a fragment thereof. Methods forpreparing and characterizing antibodies are well known in the art (See,e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988; incorporated herein by reference for the methods taught therein).

Monoclonal antibodies can be obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that can be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies can be made using the hybridomamethod first described by Kohler & Milstein, Nature 256:495 (1975), orcan be made by recombinant DNA methods (Cabilly, et al., U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas hamster can be immunized to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theantigen used for immunization. Alternatively, lymphocytes can beimmunized in vitro. Lymphocytes can be then fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,pp. 59-103 (Academic Press, 1986)).

DNA encoding a monoclonal antibody can be readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells can serve as apreferred source of such DNA. Once isolated, the DNA can be placed intoexpression vectors, which can then be transfected into host cells suchas simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences, Morrison, et al., Proc. Nat. Acad. Sci.81, 6851 (1984), or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In that manner, “chimeric” or “hybrid” antibodies can beprepared that have the binding specificity of an anti-cancer,pre-cancer, or hyperproliferative cell or other target molecule.Optionally, the antibody used herein is “humanized” or fully human.

Non-immunoglobulin polypeptides can be substituted for the constantdomains of an antibody, or they can be substituted for the variabledomains of one antigen-combining site of an antibody to create achimeric bivalent antibody comprising one antigen-combining site havingspecificity for a first antigen and another antigen-combining sitehaving specificity for a different antigen.

Chimeric or hybrid antibodies also can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature 321, 522-525 (1986); Riechmann et al., Nature 332,323-327 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies, wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

Antibodies can be humanized with retention of high affinity for thetarget site antigen and other favorable biological properties. Humanizedantibodies can be prepared by a process of analysis of the parentalsequences and various conceptual humanized products using threedimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e. theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget site antigen(s), can be achieved. In general, the CDR residuesare directly and most substantially involved in influencing antigenbinding.

Human monoclonal antibodies can be made by a hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor,J. Immunol. 133, 3001 (1984), and Brodeur, et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987).

Transgenic animals (e.g., mice) can be used that are capable, uponimmunization, of producing a repertoire of human antibodies in theabsence of endogenous immunoglobulin production. For example, it hasbeen described that the homozygous deletion of the antibody heavy chainjoining region (JH) gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. See, e.g. Jakobovits et al., Proc. Natl. Acad. Sci. USA 90,2551-255 (1993); Jakobovits et al., Nature 362, 255-258 (1993).

Alternatively, phage display technology (McCafferty et al., Nature 348,552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g. Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3, 564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature 352, 624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222, 581-597 (1991), or Griffith et al., EMBO J.12, 725-734 (1993). In a natural immune response, antibody genesaccumulate mutations at a high rate (somatic hypermutation). Some of thechanges introduced can confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as “chain shuffling”(Marks et al., Bio/Technol. 10, 779-783 (1992)). In this method, theaffinity of “primary” human antibodies obtained by phage display can beimproved by sequentially replacing the heavy and light chain V regiongenes with repertoires of naturally occurring variants (repertoires) ofV domain genes obtained from unimmunized donors. This technique allowsthe production of antibodies and antibody fragments with affinities inthe nM range. A strategy for making very large phage antibodyrepertoires (also known as “the mother-of-all libraries”) has beendescribed by Waterhouse et al., Nucl. Acids Res. 21, 2265-2266 (1993),and the isolation of a high affinity human antibody directly from suchlarge phage library is reported by Griffith et al., EMBO J. (1994). Geneshuffling can also be used to derive human antibodies from rodentantibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as “epitope imprinting,” the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT patentapplication WO 93/06213, published Apr. 1, 1993). Unlike traditionalhumanization of rodent antibodies by CDR grafting, this techniqueprovides completely human antibodies, which have no framework or CDRresidues of rodent origin.

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. One of the binding specificities is for a first antigen andthe other one is for a second antigen.

Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello, Nature 305, 537-539 (1983)). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published May 13, 1993), and inTraunecker et al., EMBO 10, 3655-3659 (1991). For further details ofgenerating bispecific antibodies see, for example, Suresh et al.,Methods in Enzymology 121, 210 (1986).

Heteroconjugate antibodies are also within the scope of the describedcompositions and methods. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Heteroconjugate antibodies can be madeusing any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

A variety of immunoassay formats can be used to select antibodies thatselectively bind with a desired target site or target site antigen. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies selectively immunoreactive with a protein, protein variant,or fragment thereof. See Harlow and Lane. Antibodies, A LaboratoryManual. Cold Spring Harbor Publications, New York, (1988), for adescription of immunoassay formats and conditions that could be used todetermine selective binding. The binding affinity of a monoclonalantibody can, for example, be determined by the Scatchard analysis ofMunson et al., Anal. Biochem., 107:220 (1980).

Not only can a targeted nanoparticle comprise an antibody or fragmentthereof, but a targeted nanoparticle can also comprise targeting ligandthat is a polypeptide or a fragment thereof. Optionally, polypeptidesthat are internalized by target cells can be attached to the surface ofa nanoparticle. Ligands that are internalized can optionally be used forinternalization of a nanoparticle into a target cell. A modified phagelibrary can be use to screen for specific polypeptide sequences that areinternalized by desired target cells. For example, using the methodsdescribed in Kelly et al., “Detection of Vascular Adhesion Molecule-1Expression Using a Novel Multimodal Nanoparticle,” Circulation Res.,(2005) 96:327-336, which is incorporated herein for the methods taughttherein, polypeptides can be selected that are internalized by VCAM-1expressing cells or other cells expressing a ligand of interest.

There are a number of methods for isolating proteins which can bind adesired target. For example, phage display libraries have been used toisolate numerous polypeptides that interact with a specific target. (Seefor example, U.S. Pat. Nos. 6,031,071; 5,824,520; 5,596,079; and5,565,332 which are herein incorporated by reference at least for theirmaterial related to phage display and methods related to combinatorialchemistry). Thus targeted nanoparticles can comprise a polypeptide orfragments thereof that interact with a desired target. A targetednanoparticle can also comprise a binding domain of an antibody or phage.

The term “polypeptide” or “peptide” is used broadly herein to mean twoor more amino acids linked by a peptide bond. The term “fragment” or“proteolytic fragment” also is used herein to refer to a product thatcan be produced by a proteolytic reaction on a polypeptide, i.e., apeptide produced upon cleavage of a peptide bond in the polypeptide. Afragment can be produced by a proteolytic reaction, but it should berecognized that a fragment need not necessarily be produced by aproteolytic reaction but can be produced using methods of chemicalsynthesis or methods of recombinant DNA technology, to produce asynthetic peptide that is equivalent to a proteolytic fragment. Itshould be recognized that the term “polypeptide” is not used herein tosuggest a particular size or number of amino acids comprising themolecule, and that a polypeptide can contain up to several amino acidresidues or more.

A nanoparticle can bind selectively or specifically to a desired targetsite, and/or can be internalized by a target cell. Such selective orspecific binding and/or internalization can be readily determined usingthe methods, systems and apparatuses described herein. For example,selective or specific binding can be determined in vivo or in vitro byadministering a targeted nanoparticle and detecting an increase in lightscattering from the nanoparticle bound to a desired target site orinternalized into the desired target cell. Detection of light scatteringcan be measured using the systems and apparatuses described below.

Thus, a targeted nanoparticle can be compared to a control nanoparticlehaving all the components of the targeted nanoparticle except thetargeting characteristics, such as, for example, targeting ligand. Bydetecting phase sensitive image data from the targeted nanoparticlebound to a desired target site versus a control nanoparticle, thespecificity or selectivity of binding or internalization can bedetermined. If an antibody, polypeptide, or fragment thereof, or othertargeting ligand is used, selective or specific binding to a target canbe determined based on standard antigen/polypeptide/epitope/antibodycomplementary binding relationships. Further, other controls can beused. For example, the specific or selective targeting of thenanoparticles can be determined by exposing targeted nanoparticles to acontrol tissue, which includes all the components of the test or subjecttissue except for the desired target ligand or epitope. To compare acontrol sample to a test sample, levels of light scattering can bedetected by, for example, the systems described below and the differencein levels or location can be compared.

A targeting ligand can be coupled to the surface or shell of at leastone of the nanoparticle. Targeted nanoparticles comprising targetingligands can be produced by methods known in the art. For exampleligands, including but not limited to, antibodies, peptides,polypeptides, or fragments thereof can be conjugated to the nanoparticlesurface.

Any method known in the art for conjugating a targeting ligand to ananoparticle can be employed, including, for example, those methodsdescribed by Hunter, et al., Nature 144:945 (1962); David, et al.,Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219(1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).Established protocols have been developed for the labeling metallicnanoparticles with a broad range of biomolecules, including protein A,avidin, streptavidin, glucose oxidase, horseradish peroxidase, and IgG(antibodies). Nanoparticles can be prepared with bioorganic molecules ontheir surface (DNA, antibodies, avidin, phospholipids, etc). Thenanoparticles can be characterized, modified, and conjugated withorganic and biomolecules. Polymers or other intermediate molecules canbe used to tether antibodies or other targeting ligands to the surfaceof nanoparticles. Methods of tethering ligands to nanoparticles areknown in the art as described in, for example, Loo et al.,“Nanoshell-Enabled Photonics-Based Imaging and Therapy of Cancer,” Tech.Cancer Res. and Treatment, (2004) 3(1) 33-40, which is incorporatedherein by reference for the methods taught herein.

Covalent binding of a targeting ligand to a nanoparticle can beachieved, for example, by direct condensation of existing side chains orby the incorporation of external bridging molecules. Many bivalent orpolyvalent agents can be useful in coupling polypeptide molecules toother particles, nanoparticles, proteins, peptides or amine functions.Examples of coupling agents are carbodiimides, diisocyanates,glutaraldehyde, diazobenzenes, and hexamethylene diamines. This list isnot intended to be exhaustive of the various coupling agents known inthe art but, rather, is exemplary of the more common coupling agentsthat can be used.

Optionally, one can first derivatize an antibody if used, and thenattach the nanoparticle to the derivatized product. As used herein, theterm “derivatize” is used to describe the chemical modification of theantibody substrate with a suitable cross-linking agent. Examples ofcross-linking agents for use in this manner include the disulfide-bondcontaining linkers SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate)and SMPT(4-succinimidyl-oxycarbonyl-α-methyl-α(2-pyridyldithio)toluene).

Targeting ligands can also be conjugated to nanoparticles using methodsincluding the preparation of biotinylated antibody molecules and theirconsequent interaction with streptavidin/nanoparticle conjugates. Thisapproach takes advantage of strong biospecific interaction betweenbiotin and streptavidin and known protocols for immobilization ofstreptavidin on nanoparticles. Polypeptides with thiol terminated alkylchains can be directly attached to the surface of nanoparticles usingthe procedures described in Elghanian, R., et al., Selectivecolorimetric detection of polynucleotides based on thedistance-dependent optical properties of gold nanoparticles. Science,1997. 277(5329): p. 1078-1080 (incorporated by reference for the methodstaught therein). For conjugation procedure one can use a mixture ofthiol terminated polypeptides and relatively small mercaptoaceticmolecules to avoid high density immobilization of the polypeptides.

Targeted nanoparticles can be prepared with a biotinylated surface andan avidinated antibody, peptide, polypeptide or fragment thereof can beattached to the nanoparticle surface using avidin-biotin bridgingchemistry. Avidinated nanoparticles can be used and a biotinylatedantibody or fragment thereof or another biotinylated targeting ligand orfragments thereof can be administered to a subject. For example, abiotinylated targeting ligand such as an antibody, protein or otherbioconjugate can be used. Thus, a biotinylated antibody, targetingligand or molecule, or fragment thereof can bind to a desired targetwithin a subject. Once bound to the desired target, the nanoparticlewith an avidinated surface can bind to the biotinylated antibody,targeting molecule, or fragment thereof. When bound in this way, lightenergy can be transmitted to the bound nanoparticle, which can producelight scattering of the transmitted light. An avidinated nanoparticlecan also be bound to a biotinylated antibody, targeting ligand ormolecule, or fragment thereof prior to administration to the subject.

When using a targeted nanoparticle with a biotinylated surface or anavidinated surface a targeting ligand can be administered to thesubject. For example, a biotinylated targeting ligand such as anantibody, polypeptide or other bioconjugate, or fragment thereof, can beadministered to a subject and allowed to accumulate at a target site

When a targeted nanoparticle with a biotinylated surface is used, anavidin linker molecule, which attaches to the biotinylated targetingligand can be administered to the subject. Then, a targeted nanoparticlewith a biotinylated shell can be administered to the subject. Thetargeted nanoparticle binds to the avidin linker molecule, which isbound to the biotinylated targeting ligand, which is itself bound to thedesired target. In this way, a three step method can be used to targetnanoparticles to a desired target. The targeting ligand can bind to allof the desired targets detailed above as would be clear to one skilledin the art.

Nanoparticles, including targeted nanoparticles, can also comprise avariety of markers, detectable moieties, or labels. Thus, for example, ananoparticle equipped with a targeting ligand attached to its surfacecan also include another detectable moiety or label. As used herein, theterm “detectable moiety” is intended to mean any suitable label,including, but not limited to, enzymes, fluorophores, biotin,chromophores, radioisotopes, colored particles, electrochemical,chemical-modifying or chemiluminescent moieties. Common fluorescentmoieties include fluorescein, cyanine dyes, coumarins, phycoerythrin,phycobiliproteins, dansyl chloride, Texas Red, and lanthanide complexes.Of course, the derivatives of these compounds are included as commonfluorescent moieties.

The detection of the detectable moiety can be direct provided that thedetectable moiety is itself detectable, such as, for example, in thecase of fluorophores. Alternatively, the detection of the detectablemoiety can be indirect. In the latter case, a second moiety reactablewith the detectable moiety, itself being directly detectable can beemployed.

A composition, including at least one nanoparticle, can be administeredto a subject orally, parenterally (e.g., intravenously), byintramuscular injection, by intraperitoneal injection, transdermally,extracorporeally, topically or the like. Parenteral administration of acomposition, if used, is generally characterized by injection.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution ofsuspension in liquid prior to injection, or as emulsions.

The compositions, including nanoparticles, can be used in combinationwith a pharmaceutically acceptable carrier. By “pharmaceuticallyacceptable” is meant a material that is not biologically or otherwiseundesirable, i.e., the material can be administered to a subject, alongwith the nanoparticle, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5.0 to about 8.0, and more preferably fromabout 7.0 to about 7.5. As described above, compositions can beadministered intravascularly. Administered compositions can includecarriers, thickeners, diluents, buffers, preservatives, surface activeagents and the like in addition to the composition of choice.Administered compositions can also include one or more activeingredients such as antimicrobial agents, anti-inflammatory agents,anesthetics, and the like.

When used in the described methods, an effective amount of one of thecompositions, including the nanoparticles, can be determined by oneskilled in the art. The specific effective dose level for any particularsubject can depend upon a variety of factors including the type andlocation of the target site, activity of the specific compositionemployed, the specific composition employed, the age, body weight,general health, sex and diet of the subject, the time of administration,the route of administration, the rate of excretion of the specificcomposition employed, the duration of the treatment, drugs used incombination or coincidental with the specific composition employed, andlike factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of the composition at levelslower than those required to achieve the desired diagnostic or imagingeffect and to gradually increase the dosage until the desired effect isachieved. If desired, an effective dose can be divided into multipledoses for purposes of administration.

Depending on the exemplary factors above, on the composition used, onthe intended target site for the composition, and whether active orpassive targeting of the described compositions is used, the timebetween administration of the described compositions and the detectionof the described nanoparticles within the subject can vary. For example,detection of the described nanoparticles can be performed at one or moretime seconds, minutes, hours, days, and/or weeks after administration ofthe compositions to the subject. When and how frequently methods ofdetection of an administered composition are performed can be determinedby one skilled in the art through routine administration and detection.

The described methods can be used to a detect cell. In some examples,the cell can be a macrophage that has engulfed a metallic particle orcomposition. The macrophage can be located anywhere in a subject, forexample, in the eye or in a vulnerable plaque. In other examples, thecell can be a cancer cell, wherein a metallic particle has been targetedto the cell. A cancer cell can be targeted anywhere in the subject. Inother examples, the cell can be any cell of a subject that has beentargeted with a metallic particle.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompositions, compositions, articles, devices, systems, and/or methodsclaimed herein are made and evaluated, and are intended to be purelyexemplary and are not intended to limit the scope of compositions,compositions, articles, devices, systems, and/or methods. Efforts havebeen made to ensure accuracy with respect to numbers (e.g., amounts,temperature, etc.), but some errors and deviations should be accountedfor. Unless indicated otherwise, parts are parts by weight, temperatureis in ° C. or is at ambient temperature, and pressure is at or nearatmospheric.

Example 1 Superparamagnetic Iron Oxide (SPIO) Nanoparticles

Colloidal suspensions of SPIO nanoparticles, Ferumoxides or AMI-25 withthe trade name Feridex® I.V. (Advanced Magnetics, Cambridge, Mass., USA)are approved by the United States Food and Drug Administration (FDA) forhuman use in 1997. The SPIO nanoparticles consisted of nonstoichiometricmagnetite cores, iron, and a dextran T-10 coating added to preventaggregation and facilitate stabilization in the liver. The mean corediameter and volume mean diameter measured by laser light scattering ofthese nanoparticles were 20 and 80 nm, respectively. Peak concentrationsof SPIO nanoparticles in the liver were observed 1 h after anintravenous injection (18 μmol Fe/kg body weight). The uptake of SPIOnanoparticles by macrophage cells is directly proportional to the IVconcentration, blood half-life, and their core size.

Experimental Preparation

As shown in FIG. 4, a schematic diagram of the apparatus is shown. Aliver sample was placed into a small rectangular plastic container (10cm×10 cm) filled with water to provide acoustic coupling between theultrasonic transducer and the specimen. The sample was imaged from thetop using a linear array transducer (128 Channel). Magnetic excitationof the sample was provided by the solenoid positioned below at thebottom surface of the water tank. The distance between the liverspecimens and iron core tip was about 1.5 mm, and the magnetic fieldstrength at this distance was measured using a tesla-meter to observethe correlation between the magnetic field strength and the ultrasoundmeasurement.

The magnetic generator comprises a solenoid (Ledex 6EC, Saia-BurgessInc., USA), a function generator (HP 33120A, Hewlett Packard Inc., USA),a current amplifier, and a regulated DC power supply. A finite elementmethod (FEM, Maxwell SV, Ansoft Inc., USA) was used to design themagnetic field generator and evaluate the magnetic flux density inresponse to a time-varying input sinusoidal current signal. The conicaliron core may be positioned close to the bottom surface of the watertank as possible while ensuring no physical contact, as to maximize themagnetic field strength applied to liver specimens. The FEM calculationsand teslameter measurements were consistent and indicated that themaximum magnetic field strength used in this experiment wasapproximately 2 T at a distance of 1.5 mm from the tip of the iron core.The FEM calculation also demonstrated that an iron core inserted in thecentre of solenoid dramatically increased and better localized themagnetic field strength at the liver specimens. Magnetic fielddistributions from the FEM simulation showed that the maximal andprincipal direction of the magnetic field strength at the liver specimenwas along the z-direction due to the conical iron core.

Ultrasound experiments were performed ex vivo using an ultrasoundimaging system (Sonosite 180 Plus, Sonosite Inc., USA) equipped with a38 mm aperture, broadband (10-5 MHz) linear array transducer. The liverspecimens were imaged in color power Doppler, power Doppler, M-mode andB-scan modes. B-scan sonogram images, also called the greyscale mode,are the typical ultrasound method to monitor or examine the human bodyusing backscattering of acoustic waves. M-mode ultrasound employs asequence of scans at a fixed ultrasound beam over a given time period.M-mode is used for visualizing rapidly moving subjects such as heartvalves. Compared to conventional B-scan images, Doppler ultrasound isused to assess changes in the frequency of reflected acoustic waves.Color power Doppler converts reflected acoustic waves that are Dopplershifted into colors that overlay the conventional B-scan images and canindicate the speed and direction of moving objects. Power Dopplerultrasound is most commonly used to evaluate moving objects and hashigher sensitivity than the color power Doppler mode. The gain of thecolor power and Doppler imaging mode may be manually adjusted tosuppress the background noise. To make objective comparisons, theexperimental settings of the ultrasound instrumentation were unchangedfor all specimens imaged.

Results

The leftmost column of FIGS. 5( a)-(e) depicts the conventional-scanimages of intact livers from mice administered 1.5, 1.0, 0.5, and 0.1(mmol Fe/kg body weight) of SPIO nanoparticles, and the controlspecimen, respectively. These images do not indicate any significantdifferences between iron-laden and control livers. In these and allother images, the ultrasound probe was positioned near the top of theliver specimens. Conventional B-scan images were obtained prior toapplying the magnetic field. In color power Doppler mode, a focusedmagnetic field (2 T) was applied. The liver with high doseadministration of SPIO (f) shows significant increasing color Dopplersignal while the normal liver (j) does not exhibit any appreciable colorDoppler signal. The M-mode signal intensity and displacement areproportional to the concentration of SPIO dose. The M-mode signalobtained from a high dose specimen (k) clearly demonstrates a sinusoidalpattern of the displacement once the magnetic field is turned on.

FIGS. 5( f)-(j) shows color power Doppler images obtained from the samelivers as depicted in FIGS. 2( a)-(e), correspondingly. These colorpower Doppler images were obtained while a 40 Hz, 20 peak-to-peak volts(Vpp) sinusoidal input was applied using a solenoid positioned below thesurface of the water tank. The peak magnetic field strength was about 2T at the specimen as measured by a tesla-meter. The field of view incolor power Doppler images (the rectangular window in FIGS. 5 (a)-(e))was centered relative to both the tip of the solenoid iron core and theliver specimens. Compared to lower dose liver specimens, the high dose(1.5 mmol Fe/kg) specimen shows progressively larger areas of Dopplersignal, thus allowing the detection of tissue-based macrophages. Nosignificant Doppler signal was observed in the control liver specimen,as shown in FIG. 5( j).

FIGS. 5( k)-(o) display M-mode measurements obtained from thecorresponding liver specimens. The horizontal X-axis in these imagesrepresents the total running time (5 s) with an ‘on-and-off’ appliedsinusoidal magnetic field (1 Hz, 20 Vpp; Bmax=1.5 T) while the Y-axisshows the vertical displacement in liver specimens, respectively. TheM-mode signal intensity and displacement at the centre position of theliver specimens increased with SPIO concentration in the liverspecimens. A high dose specimen (k) in the M-mode signal, for instance,clearly displays movement at twice the frequency of the applied magneticfield; no significant displacement was observed in the control liverspecimen (FIG. 5( o)).

FIG. 6 shows the Doppler shift from livers with different SPIO doses;1.5, 1.0, 0.5, and 0.1 mmol Fe/kg and a control liver, respectively.FIG. 6( a) shows the Doppler shift while the input signal to thesolenoid is a swept frequency ranging from 1 to 10 Hz over 1 s. FIG. 6(b)-(e) indicate a slight decrease in Doppler frequency shift withdecreasing concentration of SPIO doses in liver specimens when a sweptfrequency input signal is applied. FIG. 6( f) is the input signal of asinusoidal magnetic field at 1 Hz, 20 Vpp over 5 s. The frequencyresponse of different liver specimens was twice that of the appliedsignal; this result agrees with equation (3) and can also be observed inM-mode measurements, as shown in FIGS. 5( k)-(o). When a 1 Hz sinusoidalfrequency input signal is applied over a 5 s period, ten peak Dopplerfrequency shifts are observed over 5 s. The magnitude of the peakfrequency shift scales with SPIO dose. No significant displacement ofSPIO nanoparticles was observed in the saline-injected controlspecimens.

Example 2

FIGS. 7 a-7 f demonstrate the Doppler shift in response to an appliedmagnetic field in a liver specimen with a 1 mmol Fe/kg dose. The Dopplershift measurement used in this study was measured by the positivemaximum frequency using Matlab software (MathWorks, USA). The frequencyof the Doppler shift was exactly twice that of the modulated frequencyin all data. In all experiments, the peak Doppler shift pattern exhibitsa periodicity at exactly twice the frequency of the applied signal.

Example 3

FIG. 8( a) shows the peak Doppler frequency shift in response to a sweptfrequency input (1-10 Hz) in liver specimens with various SPIO dose. Theinset plot, as shown in FIG. 8( b), shows the peak magnetic flux densityversus voltage (Vpp: peak-to-peak volts) of the input signal. Themovement of iron-laden tissues depends directly upon the SPIO dose andstrength of the applied magnetic field. The maximum frequency shift inall specimens was observed when the magnetic field strength was 1.85 T.No significant displacement was observed in control liver specimens.

SPIO nanoparticles are identified in histological sections as bluegranules from the Prussian blue stain of control andiron-laden mouselivers, as shown in FIG. 9. Compared to the controls, FIG. 9( a), theiron-laden specimens (1.5 mmol Fe/kg bodyweight) show significantdiscrete granular iron accumulations evenly distributed in observedareas, as shown in FIG. 9( b). Although intracellular iron was alsoobserved in the control specimens, this natural iron was homogeneousrather than appearing indiscrete granular shapes. The total SPIO ironarea was 5% of the histology image as calculated by Image-Pro PLUS 5.1software (MediaCybernetics Inc., USA).

Example 4 Ferromagnetic Nanoparticles

In case of ferromagnetic nanoparticles, the frequency of the tissuedisplacement is that same as the frequency of the applied magnetic fieldand not twice as in the case of superparamagnetic nanoparticles.Ultrasound detection and methods are the same as indicated previously.Alternatively, the ferromagnetic nanoparticles have a permanent dipolemoment, and application of sound energy via ultrasound transducer mayprovide increased detection of the ferromagnetic nanoparticles viaultrasound detection.

SPIO nanoparticles taken up by macrophages in liver are detected bymagneto-motive ultrasound (MM-US) and application of a time-varyingmagnetic field produced microscale displacement of iron-laden macrophagecells. Detecting macrophages in iron-laden tissue by combining anexternal high intensity focused magnetic field provides increaseddetection by Doppler ultrasound. The physiological variation in themeasured Doppler shift, nanoparticle concentration, and other quantitiesof interest may be varied to optimize the parameters of MM-US.

The detection of magnetic nanoparticles in brain tumors using highresolution intraoperative ultrasound, and improved tumor definition inrecorded images may be possible. And the use of a B-scan image analysisusing a ‘mean grayscale’ to investigate the contrast enhancement ofsilica nanospheres (100 nm) in mice liver may be possible. Althoughthese results, which utilized B-mode images without an externallyapplied field, might hold potential for imaging brain cancer and liver,they may provide sufficient contrast enhancement for molecular imaging.The average grayscale values for the B-scan image histograms of liversamples were 41, 38, 37, 35, and 31, respectively, as shown in FIGS. 5(a)-(e)). The average brightness of the lower dose B-scan imagesdecreased slightly with lower SPIO concentration. Conversely, theultrasound reflective signal increased slightly with higher SPIO doses.Since the distinctions of grayscale values between the iron-laden andcontrol samples were not noteworthy and would depend on the clinician'sassessment, a better approach is sought after demonstrating contrastenhancement in conventional B-scan images.

To evaluate the magnetic force acting on the SPIO nanoparticles causingdisplacement of iron-laden tissue it becomes:

$\begin{matrix}{F = {{- {\nabla U}} = {{\nabla\left( {\frac{x_{s}V}{2u_{0}}{B}^{2}} \right)} = {x_{s}V{\nabla\left( \frac{{B}^{2}}{2u_{0}} \right)}}}}} & (1)\end{matrix}$

where χs, V are the susceptibility and volume of magnetic nanoparticlesand B, μ0 are the magnetic field strength and permeability,respectively. In our study, we applied a sinusoidal magnetic fieldstrength principally along the z-direction. Hence, we write {right arrowover (B)}(x, y, z; t)=sin(2Πf_(n)t)B_(z)(z){circumflex over (k)} and thetotal force F_(z) acting on the magnetic nanoparticles in thez-direction is given by

$\begin{matrix}{{\sum F_{z}} = {{m\frac{\partial^{2}{z(t)}}{\partial t^{2}}} = {F_{m} - {{kz}(t)} - {r\frac{\partial z}{\partial t}}}}} & (2) \\{{\sum F_{z}} = {\frac{x_{s}V_{s}}{2\mu_{0}}\left\lbrack {1 - {{\cos \left( {4\Pi \; f_{n}t} \right)}{B_{z}(z)}\frac{\partial B_{z}}{\partial z}} - {{kz}(t)} - {r\frac{\partial z}{\partial t}}} \right\rbrack}} & (3)\end{matrix}$

where F_(m) is magnetic force, f_(n) is the modulation frequency of theapplied sinusoidal magnetic field, −kz(t) is an elastic restoring force,and r(∂z/∂t) is a viscous drag force. Equation (3) confirmed that thefrequency response of the force acting on the SPIO nanoparticles isexactly twice the externally applied modulation frequency, f_(n).Accordingly, the frequency response of the ultrasound Doppler shift fromthe iron-laden tissue was exactly twice the modulation frequency (FIGS.5-7). The frequency doubling feature may be utilized to eliminateunwanted frequency components that arise due to environmental noise andbiological motion artifacts. The Doppler shift from tissue specimens wascorrelated with both the concentration of SPIO nanoparticles and themagnetic field strength. The concentration of SPIO nanoparticles waslinearly proportional to the Doppler shift in iron-laden macrophagecells. Increasing the magnetic field strength increases the Dopplershift of iron-laden macrophage cells (FIG. 5). In our experiment, theSPIO nanoparticles taken up by macrophages were abundant and relativelyevenly distributed in the histological images.

MM-US Application to Molecular Imaging, Diagnostics, and Treatments

The role of SPIO nanoparticles as contrast agents can be expanded fromthe diagnosis of hepatic metastases to agents that target aninflammatory response in several diseases, particularly inatherosclerosis and tumors. Macrophages are cells associated with aresponse to inflammation and degenerative disorders with high phagocyticactivity; therefore, imaging of macrophages may be useful tocharacterize diseased tissue in clinical practice. The utilization ofmagneto-motive ultrasound is applicable to intravascular ultrasound(IVUS) using a high frequency transducer by injection of smallernanoscale particles, such as ultra small SPIO (USPIO) or monocrystallineiron-oxide nanoparticles (MION), to identify macrophages as a marker ofinflammation. As shown in FIG. 10, the ultrasonic transducer can becoupled with a probe 210 for intravascular detection of diseased tissue200 associated with macrophages with nanoparticles 212. The probe can bea catheter-like endoscopic probe, which can be located within a subjectto allow sound reflection off of subject tissues or nanoparticles toobtain optical measurements, medical diagnosis, treatment, and the like.Alternatively, the probe 210 may be coupled with an Optical CoherenceTomography probe, such as a rotating catheter tip including, aturbine-type catheter as described in Patent Cooperation Treatyapplication PCT/US04/12773 filed Apr. 23, 2004 which claims priority toU.S. provisional application 60/466,215 filed Apr. 28, 2003; or arotating optical catheter tip as described in U.S. patent applicationSer. No. 11/551,684, which claims priority to U.S. provisionalapplication 60/728,481; or a rotating catheter probe as described inU.S. patent application Ser. No. 11/551,684; or an OCT-IVUS Catheter forConcurrent Luminal Imaging, U.S. provisional application 60/949,472,filed Jul. 12, 2007; each herein incorporated by reference for themethods, apparatuses and systems taught therein. The OCT probe canexpose the metallic nanoparticle to light energy whereby thenanoparticle absorbs the light energy to generate an acoustic wave thatis detected by the ultrasound transducer. Alternatively, the OCT probecan image the nanoparticle by OCT methods and systems.

The smaller USPIO and MION (hydrodynamic size: 15-30 nm) have a longerintravascular blood half-life, between 24 h and 36 h, and increasedrelative stability due to dextran coating; therefore, thesenanoparticles be taken up by macrophages in atherosclerotic vulnerableplaque and malignant tumors. Combining magneto-motive excitation withMagnetic Generator 90 with high frequency detection such as ultrasoundbiomicroscopy (UBM; 40-200 MHz operation frequency) or scanning acousticmicroscopy (SAM; 200 MHz operation frequency) may provide a dramaticincrease in resolution over conventional clinical diagnostic ultrasoundscanners. Identify vulnerable plaque and cancer cells usingmagneto-motive techniques by injection of different diameternanoparticles and dosages can be applied as indicated by parentapplications U.S. patent application Ser. No. 11/620,562, and U.S.patent application Ser. No. 11/441,824.

Finally, molecular imaging by magneto-motive ultrasound (MM-US) isvaluable for therapy treatments. A magnetic field, for example, might beused to induce magnetic hyperthermia to destroy cancer cells insurrounding tissues, as indicated in parent U.S. patent application Ser.No. 11/784,477 where the magnetic field can destroy cells by exposingthe nanoparticle to sufficient energy to heat the cell and kill thecell. Alternatively, MM-US can be coupled to a light energy source viaan OCT probe as previously described to heat the nanoparticle via lightenergy and cause photothermolysis to heat and kill the cell.

The system can further comprise an energy source for heating themetallic particle or composition. The source can provide energy forheating the composition that is sufficient to kill or lethally injurethe detected cell. In some exemplary aspects, the system can furthercomprise an energy source for causing a non-lethal change in the cell.For example, the energy source for causing a non-lethal change in thecell can produce a magnetic field. The energy source for causing anon-lethal change in the cell can also produce light or sound. Theenergy source for causing the non-lethal change in the cell and theenergy source for heating the metallic particle can be of the same type.For example, each energy source can generate and/or transmit soundenergy. In some exemplary aspects, the energy source for causing thenon-lethal change in the cell and the energy source for heating themetallic particle are the same source. In other exemplary aspects, theenergy source for causing the non-lethal change in the cell and theenergy source for heating the metallic particle are different sourcesand/or different types of energy. For example, the energy sources forcausing the non-lethal change can generates and/or transmit magneticfiled energy and the energy source for causing the heating can generateand/or transmits light energy. Thus, in some exemplary aspects thesystems described herein can comprise at least three separate sources ofenergy. One source of energy can be the magnetic energy used for MM-USimaging, as described previously. Such magnetic energy can be referredto as imaging magnetic energy. A second source can be used to produceenergy to cause heating of the metallic composition comprised by thecell to kill or lethally injure the cell. Such sources can increase thetemperature of a metallic particle in the cell. For example, any sourcethat can increase the particle temperature can be used. Exemplarysources include light energy sources and magnetic force generators thatcan cause an increase in temperature of the particle by inducingmovement of the particle. Such sources to cause lethal changes in a cellcan comprise magnetic fields, light, sound and any other energy that cancause lethal changes to a cell.

Alternatively, light energy used for the OCT imaging as would be knownto one skilled in the art. Such light energy can be referred to asimaging light energy. A second source can be used to produce energy tocauses a change in a cell. Such sources to cause changes in a cell cancomprise sources that generate magnetic fields, light, sound and anyother energy that can cause an OCT detectable change in a cell. A thirdsource of energy can be used to produce energy to cause heating of themetallic composition comprised by the cell to kill or lethally injurethe cell. Such sources can increase the temperature of a metallicparticle in the cell. For example, any source that can increase theparticle temperature can be used. Exemplary sources include light energysources and magnetic force generators that can cause an increase intemperature of the particle by inducing movement of the particle. Asdescribed above, less than there sources can be also be used. Forexample, two sources of energy can be used. In this example, lightenergy for imaging can be produced by the OCT imaging modality and cellchanging and killing energy can be generated by a second energy source,which can also be light energy.

Light energy can be generated by a light source for killing a cell. Thelight energy can be emitted over a multiplicity of optical wavelengths,frequencies, and pulse durations to achieve both OCT imaging and heatingof the nanoparticles. In one example, the heating of the nanoparticlewith light near the green spectrum can be used to cause cellular deathin the tissue targeted and localized with nanoparticles. In order toachieve heating nanoparticle and killing of the cell, the pulse durationcan be about 10 nanoseconds or less for particles smaller than 100 nm.One of skill in the art will appreciate that different pulse durationscan used for different sized nanoparticles in order to achieve heatingof the nanoparticle and cellular death. The principle of selectivephotothermolysis can be used to specify the appropriate pulse durationfor targeted particles or clusters of particles of a given size. Ifmechanical damage is to be achieved, the pulse duration can be selectedso that generated acoustic energy is confined in the particle orclusters of particles.

Advantages of Magneto-Motive Ultrasound (MM-US)

Magneto-motive ultrasound (MM-US) provides several advantages over otherimaging modalities. Since MRI utilizes static magnetic field, directapplication of this imaging approach cannot place magnetic nanoparticlesin motion. Further, since MRI is an expensive technology, enhancing thediagnostic value of ultrasound may be cost effective for obtainingimproved contrast. Second, macrophages are known to be associated withaggressive cancers of greater malignancy. Ultrasound evaluation of solidtumors is currently limited by a lack of sensitivity and specificity.Magneto-motive detection of magnetic nanoparticle-labeled macrophagesassociated with tumor cells may enhance the sensitivity and specificityof ultrasound diagnostics. For instance, ultrasound screening forprostate cancer via a rectal probe is currently a limited diagnosticmodality. The addition of a magnetic probe to an ultrasound deviceplaced over the prostate gland has the potential to improve thesensitivity and specificity of prostate cancer detection. Third, thestrong magnetic susceptibility of superparamagnetic and ferromagneticnanoparticles combined with an externally applied magnetic field is acombinatorial mechanism in biomedicine and research in targeted drugdelivery, molecular imaging, magnetic biosensing, and magneticseparation. Inasmuch as SPIO nanoparticles were approved by the FDA in1996, and are already utilized in clinical applications, many safetyconcerns of MM-US method have been addressed previously.

A novel diagnostic ultrasound imaging modality to detect SPIOnanoparticles taken up by tissue-based macrophages in a strong, highintensity magnetic field. The frequency response of the ultrasoundDoppler signal from the iron-laden tissue was twice the excitingfrequency of the input signal as predicted by magnetic force equations.MM-US provide several advantages for the diagnosis and therapy ofvarious diseases.

While the invention has been described in connection with variousembodiments, it will be understood that the invention is capable offurther modifications. This application is intended to cover anyvariations, uses or adaptations of the invention following, in general,the principles of the invention, and including such departures from thepresent disclosure as, within the known and customary practice withinthe art to which the invention pertains.

What is claimed is:
 1. An apparatus for imaging and detection,comprising: a. a magnetic field generator configured to apply a magneticfield to a cell with a plurality of metallic nanoparticles, wherein themagnetic field includes a frequency between about 1 to 10 Hz, and themagnetic field displaces the metallic nanoparticles positioned in thecell and displaces the cell; and b. an ultrasound detection systemconfigured to detect the displacement of the cell with the metallicnanoparticles displaced with the magnetic field.
 2. The apparatus ofclaim 1, wherein the ultrasound detection system is selected from thegroup consisting of color power Doppler, power Doppler, M-mode andB-scan modes.
 3. The apparatus of claim 1, wherein the ultrasounddetection system further comprises an aperture and a broadband lineararray transducer.
 4. The apparatus of claim 1, further comprising anenergy source with an energy capable of heating the detected cellthrough the metallic nanoparticles.
 5. The apparatus of claim 1, whereinthe magnetic field generator is further configured to apply a firstmagnetic field strength to the cell and apply a second magnetic fieldstrength to the cell, wherein the second magnetic field strength isdifferent from the first magnetic field strength and interacts with themetallic nanoparticles to cause a change in the cell relative tointeraction of the cell with the first magnetic field strength.
 6. Theapparatus of claim 1, wherein the magnet field generator comprises aniron-ferrite core and a solenoid coil.
 7. The apparatus of claim 1,wherein a frequency of displacement of the cell is the same as thefrequency of the applied magnetic field.
 8. The apparatus of claim 1,wherein a frequency of displacement of the cell is twice the frequencyof the applied magnetic field.
 9. A method for imaging, comprising thesteps of: a. applying a magnetic field to a cell, wherein the cellcomprises a metallic composition and the magnetic field displaces themetallic composition and the cell, wherein the magnetic field includes afrequency between about 1 to 10 Hz; and b. detecting the cell by anultrasound detection system by detecting the displacement of the cellcaused by the magnetic field with the metallic composition.
 10. Themethod of claim 9, wherein the step of applying the magnetic field tothe cell further comprises using a magnetic field generator to apply themagnetic field.
 11. The method of claim 10, wherein the magnetic fieldgenerator further comprises a solenoid, a function generator, a currentamplifier and a regulated DC power supply.
 12. The method of claim 9,wherein the metallic composition is configured to localize in the cellmembrane, in the cell cytoplasm, or on the cell surface.
 13. The methodof claim 9, wherein the step of detecting the cell further comprisesusing an ultrasound detection system selected from the group consistingof color power Doppler, power Doppler, M-mode and B-scan modes.
 14. Themethod of claim 9, wherein the cell comprises at least a macrophage, acancer cell, or a component of iron-laden tissue.
 15. The method ofclaim 9, further comprising the step of pulsing the magnetic field. 16.The method of claim 9, wherein a frequency of displacement of the cellis the same as the frequency of the applied magnetic field.
 17. Themethod of claim 9, wherein a frequency of displacement of the cell istwice the frequency of the applied magnetic field.
 18. An apparatus forimaging and detection, comprising: a. a magnetic field generatorconfigured to apply a magnetic field to a cell with a plurality ofmetallic nanoparticles, wherein the magnetic field is at least about 2T, and the magnetic field displaces the metallic nanoparticlespositioned in the cell and displaces the cell; and b. an ultrasounddetection system configured to detect the displacement of the cell withthe metallic nanoparticles displaced with the magnetic field.